GENE REGULATION BY TRASCRIPTION ANTITERMINATION

通过转录抗终止进行基因调控

• 批准号: 2391890
• 负责人: ASIS DAS
• 金额: $ 41.73万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2391890
• 关键词: DNA directed RNA polymerase; DNA footprinting; affinity chromatography; bacterial genetics; chimeric proteins; gene expression; genetic promoter element; genetic recombination; genetic regulation; genetic transcription; immunochemistry; messenger RNA; molecular cloning; nucleic acid sequence; radiation genetics; site directed mutagenesis; tissue /cell culture; transcription factor; transcription termination; translation factor

DESCRIPTION: This project continues the elucidation of fundamental mechanisms that control transcription elongation and termination in bacteria and bacteriophages. Two broad areas of work on elongation control, seeking to achieve several major objectives by combined genetic and biochemical approaches, are proposed. The first area of study, a new direction for this grant, focuses on the function and the mechanism of action of two recently discovered elongation factors of E. coli, GreA and GreB, and the function of a recently discovered collaborator (or suppressor) gene, greC. GreA and GreB are essential for cell growth at elevated temperatures. GreC mutations bypass this requirement. In vitro, GreA and GreB promote transcript cleavage by RNA polymerase (RNAP), and, by virtue of this activity, may play multiple roles in transcription: suppression of abortive initiation (and, consequently, stimulation of productive elongation and new initiation), relief from elongation arrest, and enhancement of transcription fidelity. Whether this is true in vivo is to be determined, key target genes that are regulated by GreA, GreB and GreC are to be identified, and the molecular mechanisms involved in each regulatory event are to be dissected. The second area extends ongoing studies centering around the phage l N gene protein, the archetype antiterminator that binds to a unique structure in the nascent mRNA, captures RNAP and, in conjunction with cellular proteins, transforms that RNAP to a termination-resistant state. The critical hypothesis to be tested is that the N protein and its RNA target lock RNAP into the stable elongation state by masking or modifying sites in polymerase that are critical for termination and pausing. It is proposed to identify the subunits of RNAP, and localize the sites that contact N and its collaborators: the cis-acting RNA stem-loop structure and the host factors NusA, NusG and the ribosomal protein S10. It is also proposed to localize and dissect the domains of N that recognize A, RNAP and NusA, attempt to deduce the basic principles of RNA-protein and protein-protein interactions, and illuminate the potential allosteric modifications. It further proposed to uncover the mechanisms by which N and another host factor, NusG, accelerate RNAP through pause sites and stabilize the elongation conformation, phenomena that are likely to be quintessential for termination suppression. Additional positive and negative modulators of N that might play a role in lysis-lysogeny decisions of phage development are to be isolated. A vigorous genetic hunt for a cellular homologue of N and target genes activated by an N-like antitermination mechanism will be undertaken. It is asserted that these studies should provide fundamental insights into the relationships of RNA polymerase structures with functions, and mechanisms of gene regulation.

描述：该项目继续阐明基本的 控制转录延长和终止的机制， 细菌和噬菌体。 两大工作领域对延伸率的控制，力求实现几个 主要目标的遗传和生物化学相结合的方法，是 提出了 研究的第一个领域，这项资助的新方向， 重点介绍了近年来发现的两种抗肿瘤药物的功能和作用机制， 发现了E.大肠杆菌、GreA和GreB， 最近发现的合作者（或抑制）基因的功能， greC. GreA和GreB是细胞生长所必需的。 温度 GreC突变绕过了这一要求。 体外，GreA 和GreB促进RNA聚合酶（RNAP）的转录物切割， 由于这种活性，可能在转录中发挥多种作用： 抑制流产起始（以及因此刺激 生产性伸长和新的起始），解除伸长停滞， 和增强转录保真度。 在体内是否如此 由GreA、GreB调控的关键靶基因 和GreC是要确定的，和分子机制参与， 每一个监管事件都将被剖析。 第二个领域扩展了正在进行的研究， 基因蛋白，原型抗终止子，结合到一个独特的 在新生mRNA的结构，捕获RNAP，并结合 细胞蛋白质，将RNAP转化为抗终止的 状态待检验的关键假设是N蛋白和 它的RNA靶标通过掩蔽或 修饰聚合酶中对终止至关重要的位点， 暂停。 建议鉴定RNAP的亚基，并定位 接触N及其合作者的位点：顺式作用RNA 茎环结构与宿主因子NusA、NusG和核糖体 蛋白S10。 并提出了对域进行局部化和剖分的方法 识别A，RNAP和NusA的N，试图推导出基本的 RNA-蛋白质和蛋白质-蛋白质相互作用的原理， 阐明潜在的变构修饰。 它还建议 为了揭示N和另一个宿主因子NusG， 加速RNAP通过暂停位点并稳定伸长 构象，可能是典型的现象， 终止抑制 另外的正和负调节剂 可能在噬菌体的裂解-溶原性决定中起作用的N 发展是孤立的。 一个充满活力的基因搜寻细胞 通过N样抗终止激活的N和靶基因的同源物 将采取的机制。 有人说，这些研究应该 提供了对RNA聚合酶 结构与功能，以及基因调控机制。